Development of a novel loop-mediated isothermal amplification assay for ß-lactamase gene identification using clinical isolates of Gram-negative bacteria

Front Cell Infect Microbiol. 2023 Jan 12:12:1000445. doi: 10.3389/fcimb.2022.1000445. eCollection 2022.

Abstract

Rapid evaluation of antimicrobial susceptibility is important in the treatment of nosocomial infections by Gram-negative bacteria, which increasingly carry carbapenemases and metallo-β-lactamases. We developed loop-mediated isothermal amplification (LAMP)-based assays for four β-lactamase genes (bla KPC, bla NDM-1, bla IMP-1 group, and bla VIM). The assays were evaluated using eight reference bacterial strains (Klebsiella pneumoniae, Escherichia coli, Pseudomonas aeruginosa, and Acinetobacter bereziniae) harboring six β-lactamase genes. A total of 55 Gram-negative bacterial strains, including 47 clinical P. aeruginosa isolates, fully characterized by next-generation sequencing (NGS), were used to evaluate the LAMP assays. The results were compared to those of conventional PCR. The LAMP assays were able to detect as few as 10 to 100 copies of a gene, compared to 10 to 104 copies for conventional PCR. The LAMP assay detected four β-lactamase genes with a sensitivity similar to that using purified DNA as the template in DNA-spiked urine, sputum, and blood specimens. By contrast, the sensitivity of PCR was 1- to 100-fold lower with DNA-spiked clinical specimens. Therefore, the LAMP assays were proved to be an appropriate tool for the detection of four β-lactamases.

Keywords: Gram-negative bacteria; blaIMP-1 group; blaKPC; blaNDM-1; blaVIM; loop-mediated isothermal amplification; ß-lactamase gene.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Anti-Bacterial Agents / pharmacology
  • Bacterial Proteins* / genetics
  • Escherichia coli / genetics
  • Gram-Negative Bacteria / genetics
  • Microbial Sensitivity Tests
  • Molecular Diagnostic Techniques
  • Nucleic Acid Amplification Techniques / methods
  • beta-Lactamases* / genetics

Substances

  • beta-Lactamases
  • Bacterial Proteins
  • Anti-Bacterial Agents

Supplementary concepts

  • LAMP assay

Grants and funding

DWK was supported by the grant NRF-2021R1A2C1010857 from National Research Foundation (NRF) of Korea and a grant of the Korea Health Technology R&D Project through the Korea Health Industry Development Institute (KHIDI), funded by the Ministry of Health & Welfare, Republic of Korea (grant number: HI19C0748) and Institute of Information & Communications Technology Planning & Evaluation (IITP) grant No.2020-0-01343 funded by Ministry of Science and ICT (MSIT) of Korea. EJK was supported by the grant NRF- 2020R1C1C1009992 from National Research Foundation (NRF) of Korea. MS was supported by Miyata Research Grant (A). MS and DWK were supported by Japan-Korea Basic Scientific Cooperation Program between JSPS and NRF, (grant number: JPJSBP120228812 and 2022K2A9A2A08000136). JS was supported by JSPS KAKENHI (grant number: JP22K16376). SH was supported by Nihon University Research Grant for 2022.